TY - JOUR A1 - Hartmann, Oliver A1 - Reissland, Michaela A1 - Maier, Carina R. A1 - Fischer, Thomas A1 - Prieto-Garcia, Cristian A1 - Baluapuri, Apoorva A1 - Schwarz, Jessica A1 - Schmitz, Werner A1 - Garrido-Rodriguez, Martin A1 - Pahor, Nikolett A1 - Davies, Clare C. A1 - Bassermann, Florian A1 - Orian, Amir A1 - Wolf, Elmar A1 - Schulze, Almut A1 - Calzado, Marco A. A1 - Rosenfeldt, Mathias T. A1 - Diefenbacher, Markus E. T1 - Implementation of CRISPR/Cas9 Genome Editing to Generate Murine Lung Cancer Models That Depict the Mutational Landscape of Human Disease JF - Frontiers in Cell and Developmental Biology N2 - Lung cancer is the most common cancer worldwide and the leading cause of cancer-related deaths in both men and women. Despite the development of novel therapeutic interventions, the 5-year survival rate for non-small cell lung cancer (NSCLC) patients remains low, demonstrating the necessity for novel treatments. One strategy to improve translational research is the development of surrogate models reflecting somatic mutations identified in lung cancer patients as these impact treatment responses. With the advent of CRISPR-mediated genome editing, gene deletion as well as site-directed integration of point mutations enabled us to model human malignancies in more detail than ever before. Here, we report that by using CRISPR/Cas9-mediated targeting of Trp53 and KRas, we recapitulated the classic murine NSCLC model Trp53fl/fl:lsl-KRasG12D/wt. Developing tumors were indistinguishable from Trp53fl/fl:lsl-KRasG12D/wt-derived tumors with regard to morphology, marker expression, and transcriptional profiles. We demonstrate the applicability of CRISPR for tumor modeling in vivo and ameliorating the need to use conventional genetically engineered mouse models. Furthermore, tumor onset was not only achieved in constitutive Cas9 expression but also in wild-type animals via infection of lung epithelial cells with two discrete AAVs encoding different parts of the CRISPR machinery. While conventional mouse models require extensive husbandry to integrate new genetic features allowing for gene targeting, basic molecular methods suffice to inflict the desired genetic alterations in vivo. Utilizing the CRISPR toolbox, in vivo cancer research and modeling is rapidly evolving and enables researchers to swiftly develop new, clinically relevant surrogate models for translational research. KW - non-small cell lung cancer KW - CRISPR-Cas9 KW - mouse model KW - lung cancer KW - MYC KW - JUN Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230949 SN - 2296-634X VL - 9 ER - TY - JOUR A1 - Pietro-Garcia, Christian A1 - Hartmann, Oliver A1 - Reissland, Michaela A1 - Fischer, Thomas A1 - Maier, Carina R. A1 - Rosenfeldt, Mathias A1 - Schülein-Völk, Christina A1 - Klann, Kevin A1 - Kalb, Reinhard A1 - Dikic, Ivan A1 - Münch, Christian A1 - Diefenbacher, Markus E. T1 - Inhibition of USP28 overcomes Cisplatin-resistance of squamous tumors by suppression of the Fanconi anemia pathway JF - Cell Death and Differentiation N2 - Squamous cell carcinomas (SCC) frequently have an exceptionally high mutational burden. As consequence, they rapidly develop resistance to platinum-based chemotherapy and overall survival is limited. Novel therapeutic strategies are therefore urgently required. SCC express ∆Np63, which regulates the Fanconi Anemia (FA) DNA-damage response in cancer cells, thereby contributing to chemotherapy-resistance. Here we report that the deubiquitylase USP28 is recruited to sites of DNA damage in cisplatin-treated cells. ATR phosphorylates USP28 and increases its enzymatic activity. This phosphorylation event is required to positively regulate the DNA damage repair in SCC by stabilizing ∆Np63. Knock-down or inhibition of USP28 by a specific inhibitor weakens the ability of SCC to cope with DNA damage during platin-based chemotherapy. Hence, our study presents a novel mechanism by which ∆Np63 expressing SCC can be targeted to overcome chemotherapy resistance. Limited treatment options and low response rates to chemotherapy are particularly common in patients with squamous cancer. The SCC specific transcription factor ∆Np63 enhances the expression of Fanconi Anemia genes, thereby contributing to recombinational DNA repair and Cisplatin resistance. Targeting the USP28-∆Np63 axis in SCC tones down this DNA damage response pathways, thereby sensitizing SCC cells to cisplatin treatment. KW - USP28 KW - Cisplatin KW - squamous tumors Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-273014 SN - 1476-5403 VL - 29 IS - 3 ER - TY - JOUR A1 - Prieto‐Garcia, Cristian A1 - Hartmann, Oliver A1 - Reissland, Michaela A1 - Braun, Fabian A1 - Fischer, Thomas A1 - Walz, Susanne A1 - Schülein‐Völk, Christina A1 - Eilers, Ursula A1 - Ade, Carsten P. A1 - Calzado, Marco A. A1 - Orian, Amir A1 - Maric, Hans M. A1 - Münch, Christian A1 - Rosenfeldt, Mathias A1 - Eilers, Martin A1 - Diefenbacher, Markus E. T1 - Maintaining protein stability of ∆Np63 via USP28 is required by squamous cancer cells JF - EMBO Molecular Medicine N2 - The transcription factor ∆Np63 is a master regulator of epithelial cell identity and essential for the survival of squamous cell carcinoma (SCC) of lung, head and neck, oesophagus, cervix and skin. Here, we report that the deubiquitylase USP28 stabilizes ∆Np63 and maintains elevated ∆NP63 levels in SCC by counteracting its proteasome‐mediated degradation. Impaired USP28 activity, either genetically or pharmacologically, abrogates the transcriptional identity and suppresses growth and survival of human SCC cells. CRISPR/Cas9‐engineered in vivo mouse models establish that endogenous USP28 is strictly required for both induction and maintenance of lung SCC. Our data strongly suggest that targeting ∆Np63 abundance via inhibition of USP28 is a promising strategy for the treatment of SCC tumours. KW - ∆Np63 KW - NOTCH KW - squamous cell carcinoma KW - 28 Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-218303 VL - 12 IS - 4 ER - TY - JOUR A1 - Djuzenova, Cholpon S. A1 - Fischer, Thomas A1 - Katzer, Astrid A1 - Sisario, Dmitri A1 - Korsa, Tessa A1 - Streussloff, Gudrun A1 - Sukhorukov, Vladimir L. A1 - Flentje, Michael T1 - Opposite effects of the triple target (DNA-PK/PI3K/mTOR) inhibitor PI-103 on the radiation sensitivity of glioblastoma cell lines proficient and deficient in DNA-PKcs JF - BMC Cancer N2 - Background: Radiotherapy is routinely used to combat glioblastoma (GBM). However, the treatment efficacy is often limited by the radioresistance of GBM cells. Methods: Two GBM lines MO59K and MO59J, differing in intrinsic radiosensitivity and mutational status of DNA-PK and ATM, were analyzed regarding their response to DNA-PK/PI3K/mTOR inhibition by PI-103 in combination with radiation. To this end we assessed colony-forming ability, induction and repair of DNA damage by gamma H2AX and 53BP1, expression of marker proteins, including those belonging to NHEJ and HR repair pathways, degree of apoptosis, autophagy, and cell cycle alterations. Results: We found that PI-103 radiosensitized MO59K cells but, surprisingly, it induced radiation resistance in MO59J cells. Treatment of MO59K cells with PI-103 lead to protraction of the DNA damage repair as compared to drug-free irradiated cells. In PI-103-treated and irradiated MO59J cells the foci numbers of both proteins was higher than in the drug-free samples, but a large portion of DNA damage was quickly repaired. Another cell line-specific difference includes diminished expression of p53 in MO59J cells, which was further reduced by PI-103. Additionally, PI-103-treated MO59K cells exhibited an increased expression of the apoptosis marker cleaved PARP and increased subG1 fraction. Moreover, irradiation induced a strong G2 arrest in MO59J cells (similar to 80% vs. similar to 50% in MO59K), which was, however, partially reduced in the presence of PI-103. In contrast, treatment with PI-103 increased the G2 fraction in irradiated MO59K cells. Conclusions: The triple-target inhibitor PI-103 exerted radiosensitization on MO59K cells, but, unexpectedly, caused radioresistance in the MO59J line, lacking DNA-PK. The difference is most likely due to low expression of the DNA-PK substrate p53 in MO59J cells, which was further reduced by PI-103. This led to less apoptosis as compared to drug-free MO59J cells and enhanced survival via partially abolished cell-cycle arrest. The findings suggest that the lack of DNA-PK-dependent NHEJ in MO59J line might be compensated by DNA-PK independent DSB repair via a yet unknown mechanism. KW - DNA damage KW - DNA-PK KW - Histone gamma H2AX KW - p53 KW - Radiation sensitivity Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-265826 VL - 21 ER - TY - JOUR A1 - Polat, Bülent A1 - Kaiser, Philipp A1 - Wohlleben, Gisela A1 - Gehrke, Thomas A1 - Scherzad, Agmal A1 - Scheich, Matthias A1 - Malzahn, Uwe A1 - Fischer, Thomas A1 - Vordermark, Dirk A1 - Flentje, Michael T1 - Perioperative changes in osteopontin and TGFβ1 plasma levels and their prognostic impact for radiotherapy in head and neck cancer JF - BMC Cancer N2 - Background: In head and neck cancer little is known about the kinetics of osteopontin (OPN) expression after tumor resection. In this study we evaluated the time course of OPN plasma levels before and after surgery. Methods: Between 2011 and 2013 41 consecutive head and neck cancer patients were enrolled in a prospective study (group A). At different time points plasma samples were collected: T0) before, T1) 1 day, T2) 1 week and T3) 4 weeks after surgery. Osteopontin and TGFβ1 plasma concentrations were measured with a commercial ELISA system. Data were compared to 131 head and neck cancer patients treated with primary (n = 42) or postoperative radiotherapy (n = 89; group B1 and B2). Results: A significant OPN increase was seen as early as 1 day after surgery (T0 to T1, p < 0.01). OPN levels decreased to base line 3-4 weeks after surgery. OPN values were correlated with postoperative TGFβ1 expression suggesting a relation to wound healing. Survival analysis showed a significant benefit for patients with lower OPN levels both in the primary and postoperative radiotherapy group (B1: 33 vs 11.5 months, p = 0.017, B2: median not reached vs 33.4, p = 0.031). TGFβ1 was also of prognostic significance in group B1 (33.0 vs 10.7 months, p = 0.003). Conclusions: Patients with head and neck cancer showed an increase in osteopontin plasma levels directly after surgery. Four weeks later OPN concentration decreased to pre-surgery levels. This long lasting increase was presumably associated to wound healing. Both pretherapeutic osteopontin and TGFβ1 had prognostic impact. KW - perioperative changes KW - osteopontin KW - TGFβ1 KW - head and neck cancer KW - survival Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-157529 VL - 17 IS - 6 ER - TY - JOUR A1 - Fischer, Thomas A1 - Hartmann, Oliver A1 - Reissland, Michaela A1 - Prieto-Garcia, Cristian A1 - Klann, Kevin A1 - Pahor, Nikolett A1 - Schülein-Völk, Christina A1 - Baluapuri, Apoorva A1 - Polat, Bülent A1 - Abazari, Arya A1 - Gerhard-Hartmann, Elena A1 - Kopp, Hans-Georg A1 - Essmann, Frank A1 - Rosenfeldt, Mathias A1 - Münch, Christian A1 - Flentje, Michael A1 - Diefenbacher, Markus E. T1 - PTEN mutant non-small cell lung cancer require ATM to suppress pro-apoptotic signalling and evade radiotherapy JF - Cell & Bioscience N2 - Background Despite advances in treatment of patients with non-small cell lung cancer, carriers of certain genetic alterations are prone to failure. One such factor frequently mutated, is the tumor suppressor PTEN. These tumors are supposed to be more resistant to radiation, chemo- and immunotherapy. Results We demonstrate that loss of PTEN led to altered expression of transcriptional programs which directly regulate therapy resistance, resulting in establishment of radiation resistance. While PTEN-deficient tumor cells were not dependent on DNA-PK for IR resistance nor activated ATR during IR, they showed a significant dependence for the DNA damage kinase ATM. Pharmacologic inhibition of ATM, via KU-60019 and AZD1390 at non-toxic doses, restored and even synergized with IR in PTEN-deficient human and murine NSCLC cells as well in a multicellular organotypic ex vivo tumor model. Conclusion PTEN tumors are addicted to ATM to detect and repair radiation induced DNA damage. This creates an exploitable bottleneck. At least in cellulo and ex vivo we show that low concentration of ATM inhibitor is able to synergise with IR to treat PTEN-deficient tumors in genetically well-defined IR resistant lung cancer models. KW - PTEN KW - ATM KW - IR KW - NSCLC KW - radiotherapy KW - cancer KW - DNA-PK KW - PI3K Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-299865 SN - 2045-3701 VL - 12 ER -